Molecular mechanism of Na+ -coupled HCO3- transporters: transport of CO3= and CO2

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− transporters (NCBTs, members of the SLC4 family) play critical roles in transepithelial HCO−3 Na+-coupled HCO3 transport, whole-body pH regulation, and intracellular pH (pHi) regulation. In this Multi-PI R01, the team will exploit powerful techniques, many developed in their respective laboratories, to elucidate molecular transport mechanisms of NCBTs, which are especially important in the kidney. These tools include surface pH (pHS) measurements, out-of-equilibrium (OOE) CO2/HCO3− solutions, macroscopic mathematical modeling (MMM) of acid-base transport in a single cell, and state-of-the-art molecular dynamics (MD) simulations of interactions between the substrates and the transport molecule, or of CO2 conduction through the NCBTs. In a multidisciplinary approach, the team will answer 2 major questions. Aim 1: Do all NCBTs carry some form of – ion pair—whereas other SLC4 “HCO3− ” transporters actually carry =—arriving or departing as the NaCO3 CO3 HCO3− per se? By monitoring pHS in voltage-clamped oocytes the team will test whether SLC4 family members − . They will address the same question in perfused proximal tubules (PTs) from wild- = ” vs. HCO3 transport “CO3 – binding to the KKMIK region type (WT) and NBCe1-A/D knockout mice. They also test the hypothesis that NaCO3 of TM5 is a rate-limiting step for NBCe1-A transport. Using MD, the team will identify/model outward-facing, occluded, and inward-facing conformational states of NBCe1, NBCn1 and AE1, and identify potential interaction sites –, =, −, Cl−, for NaCO3 Na+, CO3 HCO3 and and use MMM (3D reaction-diffusion simulations) to assess physiological data. Finally, in an iterative process, the team will assess single nucleotide polymorphisms (m- = ” vs. HCO−3 SNPs) as well as other mutations suggested by MD studies, prioritize them, and evaluate for “CO3 transport using physiological assay, interpret using MD and MMM, and suggest new mutations. Aim 2: Do all = ” transport, NCBTs conduct CO2 whereas other SLC4 transporters do not? Having presumably committed to “CO3 evolution faced the challenge of translocating the second carbon atom, ultimately derived from 2×HCO3−. The team will use electrophysiological techniques and a novel neutral buoyancy assay (NBA) to ask whether all NCBTs conduct CO2, whereas other SLC4 members do not. In perfused PTs from WT and NBCe1-A/D knockout mice, they will ask if NBCe1-A conducts CO2 in PTs. The team will use MD to identify potential CO2 pathways through NBCe1, NBCn1, and AE1 as a negative control. MMM will assess the physiological data. Finally, in an iterative process, the team will process m-SNPs and other mutations as outlined in Aim 1, but now for effects on CO2 conduction. The research will reorganize our thinking of NCBT function, providing valuable insight into the pathogenesis of proximal renal tubular acidosis (pRTA) and other maladies associated with NBCe1 (e.g., migraine, ocular and dental abnormalities, suicidal ideation), other NCBTs (e.g., hypertension, breast cancer, epilepsy, autism). The systematic analysis of m-SNPs may provide insights into previously unrecognized “NCBT- opathies.” The work also will have broader impact by elucidating physiological acid-base surface chemistry and = ”. − vs “CO3 for the first time permitting on to distinguish unambiguously among the transport of H+ vs HCO3